Project Summary/Abstract Protein therapeutics have played an ever-increasing role in drug discovery during the past 20 years, leading to some 380 FDA-approved drugs on the market today. All these biologics use only the tiny fraction of chemical space accessible to the standard 20 amino acids. The ability to incorporate Non-Canonical Amino Acids (NCAAs) into protein therapeutics is a promising strategy that would greatly improve the chemical sophistication of biologics, increase their bioavailability through cyclization or PEGylation, and facilitate chemical conjugation to create immunoconjugates. NCAA incorporation can be accomplished both in vitro and in vivo, but the cost of in vitro systems renders them unsuitable for production of therapeutics at scale, and cellular production is crippled by the lack of free triplet codons. I propose to harness powerful new directed evolution techniques to develop a method capable of efficiently incorporating numerous NCAAs into proteins in vivo, thereby paving the way toward low-cost, chemically-diverse protein therapeutics. Techniques in use today are limited to incorporation of no more than two NCAAs into the same protein chain. Here, I aim to improve upon this technical capability. If successful, this approach will pave the way toward production of genetically-encoded materials entirely composed of NCAAs, a technical capability that would have immediate applications for therapeutics as well as material science more broadly. I focus on frameshift suppression, an extensible technique for NCAA incorporation that offers 256 codons, rather than just two. I propose to develop a reporter for frameshift suppression, allowing me to quantify suppression efficiency more robustly than was previously possible. Next, I propose to evolve two independent molecular targets that limit frameshift suppression efficiency: suppressor tRNAs and ribosomal rRNA, in favor of improved efficiency. Existing work with traditional engineering and small libraries has seen modest success already toward the goal of improved efficiency, suggesting that my approach, which leverages a powerful continuous evolution technique, will be successful. Together, this project investigates an extensible approach to the important capability of NCAA incorporation, and aims to develop technology capable of alleviating technical difficulties that presently limit the utility of this approach.